Blind testing of routine, fully automated determination of protein structures from NMR data

The protocols currently used for protein structure determination by nuclear magnetic resonance (NMR) depend on the determination of a large number of upper distance limits for proton-proton pairs. Typically, this task is performed manually by an experienced researcher rather than automatically by using a specific computer program. To assess whether it is indeed possible to generate in a fully automated manner NMR structures adequate for deposition in the Protein Data Bank, we gathered 10 experimental data sets with unassigned nuclear Overhauser effect spectroscopy (NOESY) peak lists for various proteins of unknown structure, computed structures for each of them using different, fully automatic programs, and compared the results to each other and to the manually solved reference structures that were not available at the time the data were provided. This constitutes a stringent "blind" assessment similar to the CASP and CAPRI initiatives. This study demonstrates the feasibility of routine, fully automated protein structure determination by NMR.     

Binding of oligopeptides to d-AGATCTAGATCT and d-AAGCTTAAGCTT: can tryptophan intercalate in DNA hairpins?

The interactions of three tryptophan-containing peptides, KWK, KGWK tert-butyl ester, and KGWGK, with two self-complementary dodecamers of the same base composition but different sequence were studied by UV, CD, and fluorescence spectroscopy. The oligonucleotides, d-AGATCTAGATCT and d-AAGCTTAAGCTT, contain tandem repeats of the recognition site for the restriction enzyme BglII in the former and HindIII in the latter. Thermal transition data in dilute solutions and in 0.01 M NaCl indicate these dodecamers to be present in hairpin forms. Binding of peptides to these hairpins was followed by tryptophan fluorescence quenching titrations at 10 mM Na+; the data suggest intercalation of the indole ring. The association constants for the peptide-oligonucleotide (PN) complexes are an order of magnitude higher (10(5) M) than those reported with polynucleotides [10(4) M; Rajeswari et al. (1987) Biochemistry 26, 6825]. The pentapeptide, KGWGK, discriminates between BglII and HindIII sequences with higher affinity for the HindIII dodecamer. The CD maximum of KGWGK, at 220 nm, is drastically diminished upon interaction with oligonucleotides. The ellipticity at 220 nm is halved at 10 times less P/N ratio with the HindIII dodecamer than the BglII dodecamer, suggesting stronger binding to the HindIII dodecamer. The results are discussed in terms of two different modes of binding of oligopeptides to the DNA hairpins.     

Quantification of circulating plasma DNA in Friedreich's ataxia and spinocerebellar ataxia types 2 and 12

DNA triplet repeat expansion-associated ataxias, Friedreich's ataxia, and different types of spinocerebellar ataxias (SCAs) are progressive multisystem neurodegenerative disorders. The diagnosis of this wide group of inherited ataxias is essentially based on clinical findings. Cell-free circulating DNA in plasma has been considered as a powerful tool in clinical diagnosis and prognosis of several human diseases. In the present study, clinically suspected patients were assessed on the International Co-operative Ataxia Rating Scale and further confirmed by molecular analysis of DNA triplet repeats. Quantification of plasma DNA using a highly sensitive and DNA-specific PicoGreen fluorescent assay was done. We found significantly high levels (p?相似文献   

PPAR-Gamma as putative gene target involved in Butein mediated anti-diabetic effect

Molecular Biology Reports - Type 2 diabetes mellitus (T2DM) is a metabolic disorder caused due to varied genetic and lifestyle factors. The search for a potential natural compound to enhance the...     

Role of mannose binding lectin gene variants on its protein levels and macrophage phagocytosis with live Mycobacterium tuberculosis in pulmonary tuberculosis

Mannose-binding lectin (MBL) plays an important role in innate immunity. Functional mutant homozygotes of the MBL gene affect the serum MBL levels and have been correlated with disease susceptibility. We have studied the regulatory role of variant MBL genotypes on serum MBL level and macrophage phagocytosis with live Mycobacterium tuberculosis, and the lymphoproliferative response to M. tuberculosis culture filtrate antigen in pulmonary tuberculosis (PTB) patients (n = 48) and normal healthy subjects (NHS) (n = 58). The total serum MBL level was higher in PTB patients than in NHS (P = 0.0085). Patients and NHS with AA genotype (homozygotes of MBL - common alleles) showed a very high serum MBL level, and those with OO genotype (functional mutant homozygotes of MBL - less frequent alleles) showed a very low MBL level (AA vs. OO: NHS, P = 3.3 x 10(-9); PTB, P = 3.1 x 10(-9)). A significantly lower phagocytosis was observed in NHS with AA genotype than in NHS with AO (heterozygotes) genotype (P = 0.046). In PTB patients, no such difference was observed. A negative correlation of macrophage phagocytosis with MBL level was seen in patients and NHS (P = 0.019). Antigen-induced lymphoproliferative response was significantly decreased in PTB patients with AA genotype as compared with NHS with AA genotype (P = 0.036). The present study suggests that AA genotype with its associated higher serum MBL levels plays a regulatory role in immunity to tuberculosis than functional mutant homozygotes (OO genotype) with its associated lower level of MBL.     

Structural investigations on novel porin, OmpAb from Acinetobacter baumannii

Acinetobacter baumannii is an opportunistic pathogen and known to cause nosocomial infections especially in ICUs of hospitals. We have previously reported that the novel outer membrane protein, OmpAb from Acinetobacter baumannii is a transmembrane porin and plays an important role in transport of small molecules, like antibiotics across the membrane. In the present study we report the N-terminal sequence, Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) analysis of OmpAb and structural investigations using UV-Vis absorption, circular dichroism (CD), and fluorescence on OmpAb. SDS-PAGE results suggest that OmpAb is actually a "heat modifiable monomer" and is of 37 kDa at room temperature. Secondary structure of OmpAb is being done for the first time that showed predominantly beta-sheet structure (68%), a feature characteristic of porins. Using N-Bromosuccinimide (NBS) as oxidizing agent, the total number of tryptophans in OmpAb is estimated to be four. The present results indicate that out of the four, two tryptophans seem to be located in the integral part of the membrane, perhaps periplasmic/membrane-bound while the other two tryptophans are exposed to the solvent. We followed the fluorescence emission using conventional 280 nm and selective 305 nm excitation (established by us earlier) to explore the environment of four tryptophans in OmpAb. Emission results using selective excitation of 305 nm revealed local conformational changes of those "tryptophans which are on the surface". On urea denaturation and pH dependent denaturation there is a loss of beta-sheet structure by more than 70%, this is concomitant with the increase in fluorescence intensity and red shift in lambda(max, em). As reflected by CD spectral data, we also found that OmpAb is fairly stable like other porins up to 70 degrees C. As there are no reports on the structural aspects of any outer membrane proteins of Acinetobacter baumannii, results presented here on this novel major porin, OmpAb, will help in understanding the structure-function relationship.     

Life cycle assessment of magnetic and electronic ballast for 36-W fluorescent lamp

Background, aim, and scope   

Ballast is a device in a fluorescent lamp that supports the production of light. In this study, the environmental impacts of two types of Malaysian ballast, magnetic ballast and electronic ballast, were identified and compared using the life cycle assessment approach through the ISO 14040 (2005) series.     

In vitro polymerization of Mycobacterium leprae FtsZ OR Mycobacterium tuberculosis FtsZ is revived or abolished, respectively, by reciprocal mutation of a single residue

A single residue that dramatically influences polymerization of principal cell division protein FtsZ of Mycobacterium leprae (MlFtsZ) and Mycobacterium tuberculosis (MtFtsZ) has been identified. Soluble, recombinant MlFtsZ did not show polymerization in vitro, in contrast to MtFtsZ, which polymerised. Mutation of the lone non-conserved residue T172 in the N-terminal domain of MlFtsZ to A172, as it exists in MtFtsZ, showed dramatic polymerization of MlFtsZ-T172A in vitro. Reciprocal mutation of A172 in MtFtsZ to T172, as it exists in MlFtsZ, abolished polymerization of MtFtsZ-A172T in vitro. While T172A mutation enhanced weak GTPase activity of MlFtsZ, reciprocal A172T mutation marginally reduced GTPase activity of MtFtsZ in vitro. These observations demonstrate that the residue at position 172 plays critical role in the polymerization of MlFtsZ and MtFtsZ. A possible evolutionary correlation between the presence of polymerization-adversive or polymerization-favouring residue at position 172 in FtsZ and generation time of the respective bacterium are discussed.     

Analysis of degradation of bacterial cell division protein FtsZ by the ATP-dependent zinc-metalloprotease FtsH in vitro

The identity of protease(s), which would degrade bacterial cell division protein FtsZ in vivo, remains unknown. However, we had earlier demonstrated that Escherichia coli metalloprotease FtsH degrades E. coli cell division protein FtsZ in an ATP- and Zn(2+)-dependent manner in vitro. In this study, we examined FtsH protease-mediated degradation of FtsZ in vitro in detail using seven different deletion mutants of FtsZ as the substrates, which lack different extents of specific regions at the N- or C-terminus. FtsH protease assay in vitro on these mutants revealed that FtsH could degrade all the seven deletion mutants irrespective of the deletions or the extent of deletions at the N- or C-terminus. These observations indicated that neither the N-terminus nor the C-terminus was required for the degradation of FtsZ, like already known in the case of the FtsH substrate sigma(32) protein. The recombinant clones expressing full-length FtsZ protein and FtsZ deletion mutant proteins would be useful in investigating the possibility of FtsZ as a potential in vivo substrate for FtsH in ftsH-null cells carrying ftsH suppressor function and ectopically expressed FtsH protease.     

Burden of congenital rubella syndrome (CRS) in India based on data from cross-sectional serosurveys, 2017 and 2019–20

BackgroundIndia has set a goal to eliminate measles and rubella/Congenital Rubella Syndrome (CRS) by 2023. Towards this goal, India conducted nationwide supplementary immunization activity (SIA) with measles-rubella containing vaccine (MRCV) targeting children aged between 9 months to <15 years and established a hospital-based sentinel surveillance for CRS. Reliable data about incidence of CRS is necessary to monitor progress towards the elimination goal.MethodsWe conducted serosurveys in 2019–20 among pregnant women attending antenatal clinics of 6 hospitals, which were also sentinel sites for CRS surveillance, to estimate the prevalence of IgG antibodies against rubella. We systematically sampled 1800 women attending antenatal clinics and tested their sera for IgG antibodies against rubella. We used rubella seroprevalence data from the current survey and the survey conducted in 2017 among antenatal women from another 6 CRS surveillance sites to construct a catalytic models to estimate the incidence and burden of CRS.ResultThe seroprevalence of rubella antibodies was 82.3% (95% CI: 80.4–84.0). Rubella seropositivity did not differ by age group and educational status. Based on the constant and age-dependent force of infection models, we estimated that the annual incidence of CRS in India was 225.58 per 100,000 live births (95% CI: 217.49–232.41) and 65.47 per 100,000 live births (95% CI: 41.60–104.16) respectively. This translated to an estimated 14,520 (95% CI: 9,225–23,100) and 50,028 (95% CI: 48,234–51,543) infants with CRS every year based on age-dependent and constant force of infection models respectively.ConclusionsOur findings indicated that about one fifth of women in the reproductive age group in India were susceptible for rubella. The estimates of CRS incidence will serve as a baseline to monitor the impact of MRCV SIAs, as well progress towards the elimination goal of rubella/CRS.